When double-stranded RNA (dsRNA) is introduced into a cell, the cognate mRNA is degraded and its gene is silenced. This phenomenon is called RNA interference (RNAi) and occurs through a recently discovered biological pathway. An early step in RNAi is cleavage of the dsRNA to small pieces called siRNAs. Members of the RNase III family of enzymes are thought to catalyze this reaction, and the goal of this proposal is to understand how these enzymes participate in the process. This goal will be approached by characterizing the intrinsic biochemical properties of RNase III family members, and correlating these data with observations about RNAi made in vivo using Caenorhabditis elegans. Comparisons between wild-type animals, and animals containing deletions in each of the two C. elegans RNase III genes, will be used to correlate in vivo and in vitro data. GFP reporter constructs and immunofluorescence will be used to determine the expression patterns of the enzymes. Mutant animals, extracts prepared from these animals, and purified enzymes, will be assayed for their ability to produce siRNAs. Nucleic acid binding properties of the enzymes, and the structure of their RNA cleavage products, will be used to distinguish between mechanisms suggested by a recent crystal structure of RNase III and existing information about RNAi. Phenotypes of C. elegans strains lacking the RNase III enzymes will be characterized with an aim towards defining the roles of the enzymes in nuclear pathways. Microarray technology will be used to search for natural dsRNA substrates and their targets, and genes identified in a recent screen will be tested for nuclear functions in gene silencing. These studies will lead to information that will be helpful in using RNAi as a therapeutic agent, and also offer further insight into this recently discovered biological pathway. Understanding this biological pathway is crucial to understanding how it intersects with medically relevant pathways.